The goal of this project is to understand quinolone action in Mycobacterium tuberculosis. Studies with several bacterial species have shown that slow bacterial growth and/or cessation of growth drastically reduces the lethal activity of this class of antibacterial agent. Recent work with Escherichia coli has established the existence of a minor pathway of killing by fluoroquinolones that may bypass the negative effect of growth arrest. Exploitation of this pathway through changes in quinolone structure could, in principle, make the quinolones much more effective anti-bacterial agents. Since tuberculosis often includes a latent stage in which M. tuberculosis is "dormant", tuberculosis is expected to serve as a good model for assessing the relevance of the minor quinolone killing pathway. The present proposal focuses on the bacterial growth arrest presumed to occur shortly after infection, since in model systems growth arrest drastically reduces the activity of antimicrobial agents, including fluoroquinolones. Studies with E. coli indicate that the minor killing pathway involves the dissociation of gyrase subunits following drug-enzyme-DNA complex formation and breakage of DNA. DNA gyrase mutants have been identified that enhance this minor pathway. Identical mutants, with respect to amino acid sequence change, have been found among mycobacterial strains. These mutants will be used to determine whether enhancement of the minor lethal pathway increases the overall lethality of fluoroquinolones with M. tuberculosis during growth arrest. Relationships between gyrase mutations, quinolone structure, and lethal action are expected to provide a better understanding of the molecular interactions occurring when drug-enzyme complexes form on chromosomal DNA. Two model systems of growth arrest will be studied: low-dose aerosol infection of mice and liquid culture of M. tuberculosis under low oxygen tension. Transcription profiling of genes encoding dominant antigens will be used to assess the relevance of the in vitro system to the murine model. The study is expected to provide 1) a new view of quinolone lethality and 2) novel assays for quinolone activity. This type of work may eventually lead to new agents that effectively kill growth-arrested mycobacteria and clear infection rapidly.